Glycosphingolipids are essential components of human plasma membrane. More than 300 glycosphingolipid glycans have been characterized. The glycans are directly involved in molecular recognition event. A number of cancer-associated glycosphingolipid glycans are being developed as cancer vaccines. Most glycosphingolipid glycans are not commercially available. The costs for a limited number of commercially available ones are extremely high. We have developed highly effective one-pot multienzyme (OPME) approaches for high-yield and cost-effective production of complex glycans. The OPME approaches will be used in generating a library of 60 structurally diverse glycosphingolipid glycans including those contain different naturally occurring sialic acid forms. Three specific aims are to synthesize 1) ganglio-series; 2) lacto- and neolacto-series; and 3) globo- and isoglobo-series glycosphingolipid glycans using our established high efficient OPME strategies. Each of the 60 compounds will be synthesized under GLP conditions in 10-50 mg scale, purified to >98% purity, and characterized by nuclear magnetic resonance spectroscopy (NMR) and high-resolution mass spectrometry (HRMS). For all glycans synthesized (>98% purityL 50 micrograms of each will be sent to an NIGMS-designed screening center for validation testing, glycan microarray and other screening assays. These glycans are essential standards and invaluable probes for bioassays and biomedical applications.